Application of Ecotoxicogenomics for Studying Endocrine Disruption in Vertebrates and Invertebrates

Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans. Developing organisms are particularly sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in both reproductive and nonreproductive organs, including persistent molecular alterations. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, need to be identified for investigators to understand the molecular basis of estrogenic activity during embryonic development. For investigators to understand molecular mechanisms of toxicity in various species, toxicogenomics/ecotoxicogenomics, defined as the integration of genomics (transcriptomics, proteomics, metabolomics) into toxicology and ecotoxicology, need to be established as powerful tools for research. As the initial step toward using genomics to examine endocrine-disrupting chemicals, estrogen receptors and other steroid hormone receptors have been cloned in various species, including reptiles, amphibians, and fish, and alterations in the expression of these genes in response to chemicals were investigated. We are identifying estrogen-responsive genes in mouse reproductive tracts using cDNA microarrays and trying to establish microarray systems in the American alligator, roach, medaka, and water fleas (Daphnia magna). It is too early to define common estrogen-responsive genes in various animal species; however, toxicogenomics and ectotoxicogenomics provide powerful tools to help us understand the molecular mechanism of chemical toxicities in various animal species

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna

It is well known that most daphnid species have several attractive life history characteristics such as cyclical parthenogenesis, environmental sex determination, and predator-induced defense formation. Recent advances in high-throughput omics technologies make it easy to obtain a huge number of potential candidate factors involved in environmental stimuli-triggered phenotypic alterations. Furthermore, our group has developed a microinjection system to introduce foreign materials such as nucleotides and chemicals into the early-stage (one-cell stage) egg of Daphnia pulex and Daphnia magna. Consequently, we established a microinjection-based RNAi system that allows arbitrary gene functions to be investigated. However, this microinjection system does not seem to have pervaded in the daphnid research community due to its low throughput and high level of skills required. In this chapter, we review the microinjection method and its RNAi system in water fleas, D. pulex and D. magna, providing some technical tips and making challenging proposals for the development of novel high-throughput RNAi methods. Finally, we provide an overview of recently developed gene functional analysis methods such as overexpression and genome-editing systems

Metabolomics reveals an involvement of pantothenate for male production responding to the short-day stimulus in the water flea, Daphnia pulex

Under favorable conditions, the micro-crustacean Daphnia pulex produces female offspring by parthenogenesis, whereas under unfavorable conditions, they produce male offspring to induce sexual reproduction (environmental sex determination: ESD). We recently established a suitable system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be regulated by alterations in day-length; long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we have already demonstrated that methyl farnesoate (MF) synthesis is necessary for male offspring production, and identified ionotropic glutamate receptors as an upstream regulator of MF signaling. Despite these findings, the molecular mechanisms associated with MF signaling have not yet been well elucidated. In this study, we analyzed the whole metabolic profiles of mother daphnids reared under long-day (female-producing) and short-day (male-producing) conditions, and discovered that pantothenate (vitamin B5), a known precursor to coenzyme A, was significantly accumulated in response to the short-day condition. To confirm the innate role of pantothenate in D. pulex, this metabolite was administered to mother daphnids resulting in a significantly increased proportion of male offspring producing mothers. This study provides novel insights of the metabolic mechanisms of the ESD system in D. pulex

Dose-Related Estrogen Effects on Gene Expression in Fetal Mouse Prostate Mesenchymal Cells

Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were enriched in the glycolytic pathway. At the highest dose (100 nM), E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation

Annotation of development-dependent dimorphic genes in gonad during Day 0–12. Annotation of development-dependent significantly up- and down- regulated DEGs at FDR < 0.01 in gonadal regions incubated under MPT and FPT conditions during Day 0 to Day 12. Ordered by decreasing fold change. (XLSX 196 kb